Synthesis and biological evaluation of benzo[7,8]chromeno[5,6-b][1,4] oxazin-3-ones

Article abstract of DOI:10.1002/jhet.5570410421

The O-methylmonoximes 2,3 of stenocarpoquinone-A and β-lapachone reacted with methyl phenylacetate to give 1,4-benzoxazine derivatives 8a, 8b and oxazole 11a. Compound 8a was transformed to compounds 13_I,13 _II, 14. Treatment of compo

Full text of DOI:10.1002/jhet.5570410421

Jul-Aug 2004
605
Synthesis and Biological Evaluation of
Benzo[7,8]chromeno[5,6-b][1,4]oxazin-3-ones
#
Demetrios N. Nicolaides*a, Daman R. Gautama, Konstantinos E. Litinasa, Dimitra J.
Hadjipavlou-Litinab and Christos A. Kontogiorgisb
a
Laboratory of Organic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki,
Thessaloniki 54124, Greece.
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki,
b
Thessaloniki 54124, Greece
Received December 30, 2003
The O-methylmonoximes 2, 3 of stenocarpoquinone-A and β-lapachone reacted with methyl phenylacetate
to give 1,4-benzoxazine derivatives 8a, 8b and oxazole 11a. Compound 8a was transformed to compounds
13 , 13 , 14. Treatment of compound 14 with osmium tetroxide afforded compounds 1 5, 16 and esterification
of the latter gave the bis- and mono- esters 17 17 , 18. All products are strongly fluorescent. Compounds
I
II
I,
II
8a,b, 11a, 13-18 (azabenzo analogues of khellactones) were tested for their ability to interact with DPPH, to
compete with dimethylsulfoxide for hydroxyl radicals, to inhibit soybean lipoxygenase and trypsin activities
in vitro. Compounds 16 and 17 were found to compete significantly with dimethylsulfoxide for hydroxyl
II
radicals, whereas compounds 8a, 11a, 14 and 17 were found to inhibit strongly soybean lipoxygenase.
I
J. Heterocyclic Chem., 41, 605 (2004).
Introduction.
methanol at room temperature and separation of the reac-
tion mixture by column chromatography gave a mixture of
(Z)- and (E)-3-hydroxy-2, 2-dimethyl-3, 4-dihydro-2H-
benzo[h]chromene-5,6-dione-6-(O-methyloximes) 2I and
2II in 69% total yield (Scheme 1). The analytical data and
the recorded mass spectrum of the mixture are in good
agreement with the proposed structure of O-methyl-
monoximes 2. The 1H-nmr spectrum of the mixture
showed the presence of both isomers 2I, 2II in 1:2 ratio,
since it exhibited a pair of doublets at δ 8.02 (H-7) and
8.62 (H-7) and a pair of singlets at δ 4.26 (CH3O-) and
4.30 (CH3O-), which is a strong indication for the 1:2 pro-
portion of the isomers. The signals at δ 8.02 and 4.26 are
attributed to the (Z)- isomer 2I and the absorptions at δ
8.62 and 4.30 to the (E)- isomer 2II, since these absorp-
tions are similar to the corresponding of the H-7 and OCH3
The 2H-1,4-benzoxazine [1] structure is incorporated in
many naturally occurring substances and biologically
active compounds with anticancer [2], antibiotic [3], antitu-
mor [4] and antirheumatic [5] activities. 2H- 1 , 4 -
Benzoxazin-2-ones [6] disclosed also some interesting bio-
logical activities. These compounds were prepared by the
reaction of o-aminophenols with α-ketoesters [6] or with
dimethylacetylene dicarboxylate and triphenylphosphine
[7]. In 1996, we reported the preparation of the correspond-
ing 2H-1,4-benzoxazin-2-ones by the treatment of
O-methyl-o-quinonemonoximes with arylacetates [8],
while we recently prepared 2H-1,4-benzoxazines by the
reaction of O- m e t h y l -o-quinonemonoximes with α- b e n z y l-
ethoxycarbonylmethylene(triphenyl)phosphorane [9].
On the other hand, substituted khellactones (3',4'-
dihydroxypyranocoumarins) [10] are natural products [11]
exhibiting a broad range of biological activities, including
antifungal, antitumor, antiviral effects and activity against
H I V-1 replication [12-15]. The naturally occurring
o-quinones [16-17] stenocarpoquinone-A 1 and the lead
compound β-lapachone correlate interesting chemistry
with important pharmacological properties such as anti-
cancer activity, cytotoxic activity [18] and inhibition of
DNA topoisomerase I and reverse transcriptase [17].
In continuation of our previous efforts in the synthesis of
new coumarin derivatives [19] with potent antiinflamma-
tory activity [20], we tried to combine the 2H-1,4-benzox-
azine, khellactone and benzene (in lapachone) moieties in
order to prepare the title compounds and study their chem-
istry and biological activities as possible antiinflammatory
and antioxidant agents.
Results and Discussion.
Treatment of 3-hydroxy-β-lapachone [18] (stenocarpo-
quinone-A) 1 with methoxylamine hydrochloride in

606
D. N. Nicolaides, D. R. Gautam, K. E. Litinas, D. J. Hadjipavlou-Litina and C. A. Kontogiorgis
Vol. 41
protons of (Z)- and (E)- isomers 3I, 3II [9], which were
gradually transformed into their mixture, quick after their
separation as individual isomers.
to oxazole derivatives 11 by further methyl formate radical
abstraction. In a recent paper, the high yield transforma-
tion of benzoxazinones to the corresponding benzoxazoles
have been reported by the treatment with 10% potassium
hydroxide in refluxing methanol [26]. The analytical and
spectral data of the products resemble well the structures
8a, 8b, 11a suggested for them.
Treatment of 2(I+II) with methyl phenylacetate at 180 °C
for 30 minutes and separation of the reaction mixture by
column chromatography afforded 6,6-dimethyl-2-phenyl-
5,6-dihydro-4H-benzo[7, 8]chromeno[6, 5-d][1,3]oxazol-
5-ol 11a (11%) and 6-hydroxy-7,7-dimethyl-2-phenyl-6,7-
dihydro-3H,5 H-benzo[7,8]chromeno[5,6-b][1,4]oxazin-3-
one 8a (37%). When the reaction was repeated in a higher
scale compounds 11a, 8a were obtained in 9% and 44%
yields respectively.
By a similar treatment of 3(I+II) with methyl phenyl-
acetate for 1 hour and separation of the complicated reac-
tion mixture by column chromatography, only compound
8b was obtained in 21% yield. Compound 11b was not
detected or separated from the reaction mixture.
Compounds 8, 11 can be obviously formed through the
mechanism suggested in Scheme 2. The reaction between
the initially formed radicals 4 and 5 through 6 followed by
methanol elimination [21] can lead to the (Z)- and/or (E)-
intermediate 7. Lactonization of the (Z)- isomer only pro-
vides [8] compounds 8 a , b. Intramolecular nucleophilic
attack of hydroxyl to the imine moiety [8,21-24] can lead
to the intermediate 9. Hydrogen abstraction by the former
radicals 4, 5 present [25] lead to the radical 10 and finally
Treatment of compound 8a with (1S)-(-)-camphanic
chloride 12 in dry pyridine/dichloromethane at 0 °C under
n i t r o g e n atmosphere gave (Scheme 3) a mixture of
diastereoisomers 7,7-dimethyl-3-oxo-2-phenyl-6,7-dihy-
dro-3H,5H-benzo[7,8]chromeno[5,6-b][1,4]oxazin-6-yl
4, 7, 7-trimethyl-3-oxo-2-oxabicyclo[2. 2. 1]heptane-1-
carboxylates 13I, 13II in 60% yield followed by the pure
diastereomer 13II (11%), which exhibited signals in its 1H-
nmr spectrum for camphanic CH3 at δ 0.88, 0.93, 1.06, and
finally unreacted compound 8a (23%). Efforts for the sep-
aration of the pure stereoisomer 13I by repeated chromato-
graphic separations were not successful. The 1H-nmr spec-
trum of the above mixture exhibited absorptions for the
camphanic CH3 at δ 0.92, 1.01, 1.07 and 0.88, 0.93, 1.06
in 1:2 ratio. It means that the mixture constituted from 13I
and 13II in 1:2 ratio and the total yield of 13II was 51%.
Treatment of compound 8 a with triphenylphosphine in
dry carbon tetrachloride/acetonitrile solution [27] under
reflux gave 7,7-dimethyl-2-phenyl-3H, 7H-benzo[7, 8]-

Jul-Aug 2004
Synthesis and Biological Evaluation of Benzo[7,8]chromeno[5,6-b][1,4]oxazin-3-ones
607
chromeno[5, 6- b][1,4]oxazin-3-one 14 in 88% yield.
Treatment of compound 14 with osmium tetroxide/N-
methylmorpholine N-oxide [28] in acetone-water at
room temperature and separation of the reaction mixture
by column chromatography afforded 6-hydroxy-7,7-
d i m e t h y l - 2 - p h e n y l - 6 , 7 - d i h y d r o - 3 H, 5H- b e n z o [ 7 , 8 ]-
chromeno[5, 6-b][1,4]oxazine-3,5-dione 15 (23%) and
5 , 6 - d i h y d r o x y - 7 , 7 - d i m e t h y l - 2 - p h e n y l - 6 , 7 - d i h y d r o -
3H, 5H-benzo[7, 8]chromeno[5, 6-b][1, 4]oxazine-3-one
16 (54%) together with 6% unreacted starting compound
14. The 6-hydroxy-structure 15 instead of the 5-hydroxy
structure was suggested on the basis of the comparison
of the 1H-nmr spectrum of the compound and especially
of the signals of its 7,7-dimethyl protons at δ 1.40 and
1.86 with those of compounds 8a (at δ 1.43 and 1.49),
11a (1.43 and 1.58), 2I (1.47), 2II (1.41), 16 (1.57) and
other compounds [29], since the corresponding two
methyl proton signals of ethyl 2,2-dimethyl-3,6-dioxo-
3, 4-dihydro-2 H, 6H-benzo[f]pyrano[2, 3- h]chromene-8-
carboxylate [30] exhibited signals at δ 1.59 (6H). The
formation of compound 15 can be explained by partial
oxidation of the dihydroxy derivative 16 with osmium
tetroxide [30,31].
Treatment of compound 16 with chloride 12 in dry
pyridine/carbon tetrachloride at room temperature and
separation of the reaction mixture by column chroma-
tography gave the two isomers (5R, 6R)- and (5S, 6S) - d i-
O-(-)camphanoyl diastereoisomers (cis)-7,7-dimethyl-3-
oxo-2-phenyl-5-{[(4,7,7-trimethyl-3-oxo-2-oxabicyclo-
[ 2 . 2 . 1 ] h e p t - 1 - y l ) c a r b o n y l ] o x y } - 6 , 7 - d i h y d r o - 3 H, 5H-
b e n z o [ 7 , 8 ] c h r o m e n o [ 5 , 6 - b][1,4]oxazin-6-yl 4,7,7-
trimethyl-3-oxo-2-oxabicyclo[2. 2. 1]heptane-1-carbo-
xylates 17I (9%) and 17II (12.5%) and 6-hydroxy-7,7-
dimethyl-3-oxo-2-phenyl-6, 7-dihydro-3H, 5H-benzo-
[7,8]chromeno[5,6-b][1,4]oxazin-5-yl 4,7,7-trimethyl-3-
oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate 18, in
22% yield. It was not able to assign the absolute R,R or
S,S configuration at the C-5 and C-6 of compounds 17I,
17II on the basis of the data available. We suggest the 6-
hydroxy-structure 18 for the monoester in question
instead of the also possible 5-hydroxy peri-isomer struc-
ture on the basis of the comparison of its recorded signals

608
D. N. Nicolaides, D. R. Gautam, K. E. Litinas, D. J. Hadjipavlou-Litina and C. A. Kontogiorgis
Vol. 41
change of absorbance produced in this reaction is assessed
to evaluate the antioxidant potential of test samples and
this assay is useful as a primary screening system. All
compounds, comparing to acetylsalicylic acid (80.5 %) or
nor-dihydroguaeretic acid (94.4 %) used as standard drugs,
were found to interact (16.7-80.8 %) with DPPH. For com-
pounds 8b, 14, 15 and 17I the interaction increases with
the increase of the concentration of the tested compounds,
whereas compound 17II highly interacts (100%) in both
concentrations.
Because hydroxyl radicals is one of the most potent oxi-
dizing agents which under certain conditions might be
implicated in lipid peroxidation and because hydrogen per-
oxide as a source of hydroxyl radicals has been implicated
in inflammation [34], we attempted to investigate the abil-
ity of the synthesized compounds to compete with
dimethylsulfoxide for hydroxyl radicals. The competition
of compound 11a for hydroxyl radicals was very low (6.7
%), whereas for compounds 15, 16 and 17II was higher
(23.6 %, 40.3 % and 46.6%). Compounds 8a-b, 13II, and
14 did not show any effect under the reported experimental
conditions for dissolution reasons.
at δ 4.27 (d, 6-H) and 6.62 (d, 5-H), since the signals for
the compounds 16, 17I, 17II were observed at δ 4.00,
5.63, 5.54 for 6-H and at 5.33, 6.84, 6.78 for 5-H
respectively.
All the title compounds prepared show a very strong
fluorescence.
Biological Evaluation.
The compounds were screened for biological activities
(Table 1) by in vitro assays.
Inhibitory activities were measured against isolated
enzymes (soybean lipoxygenase and trypsin). A qualita-
tive similarity between the inhibition of plant lipoxygenase
activity by non-steroid anti-inflammatory drugs and the
corresponding inhibition of the mammalian mast cells
lipoxygenase has been reported [32]. Monohydroxy
coumarins e.g. 7-OH, inhibited the formation of 5-lipoxy-
genase. Compounds 8b, 11a, 14 and 17I were found to
highly inhibit soybean lipoxygenase. The role played by
proteases in the early stage of inflammatory process is well
documented. Some anti-inflammatory compounds have
been reported to inhibit trypsin. Only the 17II isomer
exhibits significant inhibition.
The reducing abilities of the examined compounds were
determined by their interaction with the stable free radical
1,1-diphenylpicrylhydrazyl (DPPH). Antioxidants can
react with DPPH producing 1,1-diphenylpicrylhydrazine
[33]. Due to its odd electron DPPH gives a strong absorp-
tion band at 517 nm. As this electron becomes paired off in
the presence of a free radical scavenger, the absorption
vanishes and the resulting decolorization is stoichiometric
with respect to the number of electrons taken up. The
Compounds 8a, 14 and 16 seemed to be highly cytotoxic
in an anti-HIV screening test (results not shown).
Lipophilicity in our case does not seem to affect the bio-
logical activity. On the contrary sterimol parameters,
expressing steric requirements must be more important.
Certain similarities in the biological screening exist among
the 8a, 11a, 14 and 16 structures, whereas the 17I isomer
combines good antioxidant properties in correlation to its
significant inhibition of lipoxygenase. It should be possi-
ble that no specificity exists in the presence of an 1,4-
oxazine or 1,3-oxazole ring fused to naphthopyran skele-
ton, on the contrary the presence of the naphthopyran moi-
ety seems to be crucial for the biological activity.
Table 1
Reduction Ability[a] (RA %), Competition with Dimethylsulfoxide for
.
Hydroxyl Radical [b] (HO %), Soybean Lipoxygenase Inhibition [c]
(LOX %), Trypsin Inhibition [d] (Itr %)
EXPERIMENTAL
.
Compound RA %
0.1mM
HO %
1mM
(LOX %)
0.1mM
(Itr %)
0.1mM
0.5mM
Melting points are uncorrected and were measured on a Kofler
hot-stage apparatus. IR spectra were obtained with a Perkin-
Elmer 1310 spectrophotometer as nujol mulls. Nmr spectra were
recorded on a Bruker AM 300 (300 MHz, and 75 MHz), for H
13
and C respectively, using deuteriochloroform as solvent and
tetramethylsilane as an internal standard; J values are reported in
Hz. Mass spectra were determined on a VG-250 spectrometer at
70 eV under Electron Impact (EI) conditions, or on a Perkin
Elmer API 100 Sciex Simple quadrupole under Electronspray
Ionization (ESI) conditions. High resolution mass spectra (hrms)
were recorded on an Ionspec mass spectrometer under Matrix-
Assisted Laser Desorption-Ionization Fourier Transform Mass
Spectrometer (MALDI-FTMS) conditions with 2,5-dihydroxy-
benzoic acid (DHB) as the matrix. Microanalyses were per-
formed on a Perkin-Elmer 2400-II Element analyzer. Analyses
indicated by the symbols of the elements were within ±0.4% of
the theoretical values. Earlier reported procedures were used for
8a
8b
11a
13
14
15
16
17
17
18
24.8
16.7
25
40.4
24.8
12.3
30.1
80.8
100
20
24.1
36.5
25
40.4
26
36
31
100
100
21
No
No
6.7
No
No
23.6
40.3
No
46.6
Nt
No
71.4
79.2
Nt
80.2
No
Nt
51.7
No
No
No
Nt
Nt
No
2
Nt
38.2
6
1
II
I
II
No
No
Nt: not tested; No: no action under the reported experimental conditions-
dissolution problems; Data are means of two or three independent exper-
iments and the deviation in absorbance values were less than 10 %; [a]
Acetyl-salicylic acid as a standard 80.5 % (0.1 mM) and nor-dihydro-
guaeretic acid 94.4 %; [b] tocopherol acetate as a standard 83.4 %,
dimethylsulfoxide as a standard 78.5 %; [c] nor–dihydroguaeretic acid
83.7 % (0.1 mM); [d] salicylic acid 18.1 % (0.1 mM).
the preparation of compounds 1 [18] and 3 3 [9].
I, II

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Synthesis and Biological Evaluation of Benzo[7,8]chromeno[5,6-b][1,4]oxazin-3-ones
609
Preparation of (Z)- and (E)-3-Hydroxy-2,2-dimethyl-3,4-dihy-
d r o - 2 H- b e n z o [ h] c h r o m e n e - 5 , 6 - d i o n e - 6 - (O-methyloximes)
2 + 2 .
~180 °C for 1 hour and then was concentrated in a rotary evapo-
rator. The residue was separated by column chromatography (sil-
ica gel, hexane/ethyl acetate 100:1) to give yellow crystals of
compound 8 b (34 mg, 21%), m.p. 220-222°C (from
I
II
A solution of 3-hydroxy-β-lapachone 1 (1.0 g, 3.876 mmoles)
-1
1
ether/hexane); ir: 3050, 1725, 1595 cm ; H-nmr: δ 1.50 (s, 6H),
1.99 (t, 2H, J=6.4), 3.00 (t, 2H, J=6.4), 7.50-7.58 (m, 4H), 7.68
(t, 1H, J=7.6), 8.23 (d, 1H, J=7.6), 8.49-8.52 (m, 2H), 8.79 (d,
and methoxylamine hydrochloride (324 mg, 3.879 mmoles) in
methanol (75 mL) was stirred at room temperature for 2 hours,
until full consumption of quinone. The solvent was evaporated in
a rotary evaporator and the residue was subjected to a column
chromatography (silica gel, hexane/ethyl acetate 2:1) to give a
yellow solid, (1:2) mixture of isomers 2 and 2 (0.77 g, 69%)
13
1H, J=7.6); C-nmr: δ 16.5, 26.7, 31.5, 76.5, 104.5, 121.8,
122.4, 123.6, 125.8, 128.0, 128.2, 128.9, 129.7, 130.2, 134.9,
+
135.1, 143.1, 145.3, 153.3, 159.3; ms (ESI): m/z 358 (M+H) ,
+
380 (M+Na) ; For C
+
NO hrms Calcd. 358.1443 (MH );
I
II
H
23 20
3
m.p. 180-183°C (from ether); ir: 3430, 3390, 3070, 1630, 1605
Found 358.1426.
Anal. Calcd for C
-1
1
cm ; H-nmr: δ 1.41 (s, 12H), 1.47 (s, 6H), 2.04 (brs, 3H), 2.56-
2.66 (m, 3H), 2.78-2.86 (m, 3H), 3.87 (t, J=5.09, 3H), 4.26 (s,
3H), 4.30 (s, 6H), 7.42-7.47 (m, 6H), 7.83 (d, J=8.9, 1H), 7.92 (d,
H
NO : C, 77.29; H, 5.36; N, 3.92.
23 19 3
Found: C, 77.51; H, 5.43; N, 3.91.
13
Reaction of Compound 8a with (1S)-(-)-Camphanic Chloride 12.
J=6.4, 2H), 8.02 (d, J=6.4, 1H), 8.62 (d, J=6.4, 2H); C-nmr: δ
21.8, 21.9, 24.9, 25.5, 25.7, 64.9, 68.7, 80.1, 80.3, 108.8, 113.4,
123.3, 123.7, 123.8, 126.4, 128.1, 128.8, 129.1, 129.8, 130.0,
A solution of compound 8a (51 mg, 0.136 mmole) and com-
pound 1 2 (122 mg, 0.563 mmole) in a dry mixture of
dichloromethane (4 mL) and pyridine (1 mL) was stirred at room
temperature for 16 hours. The mixture was then concentrated in a
rotary evaporator and the residue was treated with water (10 mL)
and the mixture was extracted with chloroform (10 mL). The
o rganic layer was washed with water (5 mL) and brine (5 mL),
dried (sodium sulfate) and concentrated in a rotary evaporator.
The residue was subjected to column chromatography (silica gel,
dichloromethane/ethyl acetate 50:1). The fractions eluted first
+
130.2, 130.6, 130.8, 144.4, 182.7; ms (ESI): m/z 288 (M+H) ,
+
310 (M+Na) .
Anal. Calcd for C H NO : C, 66.88; H, 5.96; N, 4.88.
16 17 4
Found: C, 67.13; H, 5.80; N, 4.81.
Reaction of Monoximes 2 with Methyl Phenylacetate.
(I+II)
A: A mixture of compounds 2
(150 mg, 0.52 mmole) and
(I+II)
methyl phenylacetate (0.9 mL, excess) was heated in an oil bath
at ~175-180 °C for 30 minutes and then it was concentrated in a
rotary evaporator. The residue was subjected to a column chro-
matography (silica gel, hexane/ethyl acetate 12:1). The fractions
eluted first gave compound 11a (19 mg, 11%) as colorless crys-
tals, m.p. 271-272 °C (from ethyl acetate/hexane); ir: 3390, 3050,
gave a yellow solid of a mixture of diastereoisomers 13 13 (45
I,
II
1
mg, 60%), with H-nmr: δ 0.88 (s, 6H), 0.92 (s, 3H), 0.93 (s, 6H),
1.01 (s, 3H), 1.06 (s, 6H), 1.07 (s, 3H), 1.51 (s, 6H), 1.53 (s, 12H),
1.56-1.74 (m, 3H), 1.81-1.95 (m, 3H), 1.96-2.14 (m, 3H), 2.30-
2.45 (m, 3H), 3.06 (dd, 1H, J =5.5, J =17.3), 3.12 (dd, 2H,
1
2
J =5.5, J =17.3), 3.43 (dd, 3H, J =5.5, J =17.3), 5.35 (t, 3H,
-1
1
1570 cm ; H-nmr: δ 1.43 (s, 3H), 1.58 (s, 3H), 1.79 (brs, 1H,
exchanged with deuterium oxide), 3.17 (dd, 1H, J =3. 81,
J =17.8), 3.35 (dd, 1H, J =5.1, J =17.8), 4.04 (dd, 1H, J =3.81,
1
2
1
2
1
J=5.5), 7.42 (t, 1H, J=7.6), 7.51-7.55 (m, 9H), 7.60 (t, 2H, J=8.2),
7.74 (t, 2H, J=8.2), 7.89 (t, 1H, J=7.6), 8.26 (d, 2H, J=8.2), 8.33
(d, 1H, J=7.6), 8.48-8.51 (m, 6H), 8.85 (d, 3H, J=8.2). The frac-
tions eluted next gave yellow crystals of compound 13 (8 mg,
II
11%, total 38 mg, 51%), m.p. 268-270°C (from ether/hexane); ir:
-1 1
1785, 1730, 1690, 1580 cm ; H-nmr: δ 0.88 (s, 3H), 0.93 (s, 3H),
2
1
2
1
J =5.1), 7.46-7.55 (m, 4H), 7.65 (t, 1H, J=7.6), 8.20 (d, 1H,
13
2
J=7.6), 8.24-8.27 (m, 2H), 8.50 (d, 1H, J=7.6); C-nmr: δ 22.8,
24.4, 26.6, 68.7, 78.0, 106.6, 122.0, 122.8, 123.6, 124.7, 127.0,
127.1, 127.5, 128.8, 130.5, 130.6, 155.9, 158.5, 159.6, 160.9; ms
+
(ESI): m/z 346 (M+H) .
Anal. Calcd for C H NO : C, 76.50; H, 5.55; N, 4.06.
22 19 3
Found: C, 76.68; H, 5.42; N, 4.07.
1.06 (s, 3H) 1.51 (s, 3H), 1.53 (s, 3H), 1.56-1.74 (m, 1H), 1.81-
1.95 (m, 1H), 1.96-2.14 (m, 1H), 2.30-2.45 (m, 1H), 3.12 (dd, 1H,
J =5.5, J =17.3), 3.43 (dd, 1H, J =5.5, J =17.3), 5.35 (t, 1H,
1
2
1
2
The next fractions gave compound 8a (72 mg, 37%), as yellow
crystals (green fluoresence), m.p. 271-273°C (from ethyl
J=5.5), 7.51-7.55 (m, 3H), 7.60 (t, 1H, J=8.2), 7.74 (t, 1H, J=8.2),
13
8.26 (d, 1H, J=8.2), 8.48-8.51 (m, 2H), 8.85 (d, 1H, J=8.2); C-
-1
1
acetate/hexane); ir: 3450, 3060, 1730, 1580 cm ; H-nmr: δ
1.45 (s, 3H), 1.51 (s, 3H), 1.60 (brs, 1H, exchanged with deu-
terium oxide), 3.07 (dd, 1H, J =5.1, J =17.8), 3.24 (dd, 1H,
nmr: δ 9.6, 16.7, 22.7, 23.1, 24.9, 28.9, 30.8, 54.1, 54.7, 71.2,
90.7, 101.7, 122.0, 122.6, 123.2, 126.2, 126.9, 128.3, 128.5,
129.0, 129.9, 130.4, 130.6, 132.8, 140.6, 144.2, 148.0, 157.5,
1
2
+
J =5.1, J =17.8), 4.03 (t, 1H, J=5.1), 7.52-7.62 (m, 4H), 7.72 (t,
159.0, 177.7, 180.3; ms (EI): m/z 553 (35, M ), 525 (10), 355
(20), 312 (75), 273 (70), 105 (100), 77 (65); For C H NO hrms
1
1H, J=7.6), 8.25 (d, 1H, J=7.6), 8.47-8.51 (m, 2H), 8.82 (d, 1H,
2
33 32
Calcd. 554.2173 (MH ); Found 554.2155. Unreacted compound
7
13
J=7.6); C-nmr: δ 22.3, 24.7, 25.8, 68.5, 79.0, 102.6, 122.0,
122.6, 123.3, 126.1, 128.2, 128.3, 129.1, 130.5, 130.6, 135.0,
+
139.8, 153.8, 155.2, 160.0, 161.3; ms (ESI): m/z 374 (M+H) ,
+
396 (M+Na) .
+
8a was eluted next (12 mg, 23%).
Dehydration of Compound 8a. Preparation of Compound 14.
Anal. Calcd for C H NO : C, 73.98; H, 5.13; 3.75. Found:
4
A solution of compound 8a (314 mg, 0.84 mmole) and triph-
enylphosphine (655 mg, 2.49 mmoles) in a mixture of carbon
tetrachloride (11 mL) and acetonitrile (11 mL) was heated under
reflux for 2.5 hours. The mixture was concentrated in a rotary
evaporator and the residue was subjected to column chromatog-
raphy (silica gel, hexane/ethyl acetate 25:1) to give yellow crys-
tals of compound 14 (0.261 g, 88%), m.p. 213-215°C (from ethyl
23 19
C, 73.88; H, 4.89; N, 3.74.
B: When the reaction was repeated between compounds 2
(I+II)
(550 mg, 1.916 mmoles) and methyl phenylacetate (2.75 mL,
excess) compounds 11a (9%) and 8a (44%) were obtained again.
Reaction of Monoximes 3
with Methyl Phenylacetate.
(I+II)
-1
1
acetate/hexane); ir: 1710, 1585 cm ; H-nmr: δ 1.61 (s, 6H), 5.82
(d, 1H, J=10.2), 6.95 (d, 1H, J=10.2), 7.52-7.59 (m, 4H), 7.69 (t,
A mixture of compounds 3 (121 mg, 0.446 mmole) and
(I+II)
methyl phenylacetate (4 mL, excess) was heated in an oil bath at

610
D. N. Nicolaides, D. R. Gautam, K. E. Litinas, D. J. Hadjipavlou-Litina and C. A. Kontogiorgis
Vol. 41
1H, J=7.6), 8.22 (d, 1H, J=8.9), 8.49-8.53 (m, 2H), 8.79 (d, 1H,
13
J=7.6); C-nmr: δ 28.2, 78.7, 105.0, 115.0, 121.1, 122.1, 122.7,
122.9, 126.0, 128.3, 128.6, 128.9, 129.7, 130.4, 134.9, 142.1,
+
143.8, 152.3, 153.0, 159.0; ms (ESI): m/z 356 (M+H) , 378
+
(M+Na) .
Anal. Calcd for C H NO : C, 77.73; H, 4.82; N, 3.94.
23 17 3
Found: C, 77.55; H, 4.72; N, 3.88.
1.99 (m, 3H), 2.04-2.22 (m, 3H), 2.27-2.44 (m, 1H), 2.51-2.64
(m, 1H), 5.63 (d, 1H, J=3.8), 6.84 (d, 1H, J=3.8), 7.50-7.53 (m,
3H), 7.63-7.80 (m, 2H), 8.28 (d, 1H, J=8.9), 8.42-8.50 (m, 2H),
+
8.86 (d, 1H, J=7.6); ms (EI): m/z 749 (5, M ), 170 (40), 152 (25),
139 (80), 125 (40), 109 (99), 83 (85), 55 (100); For
C
+
NO Na hrms Calcd. 772.2728 (MNa ); Found 772.2742.
H
43 43
The fractions eluted next gave yellow crystals of compound
11
17 (22 mg, 12.5%), m.p. 212-214 °C (from ether/hexane); ir
II
(dichloromethane): 3035, 1780, 1745, 1725, 1690, 1675, 1570
Reaction of Compound 14 with Osmium Tetroxide. Preparation
of Compounds 15 and 16.
-1
1
cm ; H-nmr: δ 1.00 (s, 3H), 1.01 (s, 3H), 1.08 (s, 3H), 1.11 (s,
3H), 1.13 (s, 3H), 1.57 (s, 6H), 1.62 (s, 3H), 1.60-1.78 (m, 2H),
1.85-2.00 (m, 2H), 2.21-2.33 (m, 2H), 2.45-2.59 (m, 2H), 5.54 (d,
1H, J=5.08), 6.78 (d, 1H, J=5.08), 7.45-7.58 (m, 3H), 7.63 (t, 1H,
J=7.6), 7.80 (t, 1H, J=7.6), 8.28 (d, 1H, J=8.9), 8.42-8.50 (m,
To a solution of compound 14 (200 mg, 0.56 mmole) and N-
methylmorpholine N-oxide (133 mg, 1.13 mmoles) in 80% aque-
ous acetone (28 mL, free from ethanol) an 4% aqueous solution
of osmium tetroxide (0.46 mL) was added and the mixture was
stirred at room temperature for 18 hours. Water (15 mL) was then
added and the mixture was concentrated in a rotary evaporator to
an aqueous residue, which was extracted with ethyl acetate (3x15
mL). The organic layer was dried over anhydrous sodium sulfate,
the solvent was evaporated and the residue was separated by col-
umn chromatography (silica gel, hexane/ethyl acetate 6:1). The
fractions eluted first gave yellow crystals of compound 15 (50
mg, 23%), m.p. 214-216 °C (from ethyl acetate/hexane); ir: 3460,
+
2H), 8.87 (d, 1H, J=7.6); ms (EI): m/z 749 (25, M ), 326 (10),
170 (28), 139 (60), 125 (30), 109 (100), 83 (65); For
+
NO Na hrms Calcd. 772.2728 (MNa ); Found 772.2738.
C
H
43 43
11
The following fractions afforded yellow crystals of compound
18 (29 mg, 22%), m.p. 226-228 °C (from ether/hexane); ir: 3430,
-1
1
3040, 1780, 1730, 1690, 1600, 1580 cm ; H-nmr: δ 1.02 (s,
3H), 1.05 (s, 3H), 1.09 (s, 3H), 1.66 (s, 6H), 1.85-1.97 (m, 2H),
2.08-2.21 (m, 1H), 2.41-2.58 (m, 1H), 2.72 (brs, 1H), 4.25 (d, 1H,
J=5.1), 6.62 (d, 1H, J=5.1), 7.48-7.52 (m, 3H), 7.60 (t, 1H,
J=7.6), 7.76 (t, 1H, J=7.6), 8.28 (d, 1H, J=7.6), 8.43-8.47 (m,
-1
1
3040, 1685, 1675, 1625, 1590 cm ; H-nmr: δ 1.40 (s, 3H), 1.86
(s, 3H), 3.94 (s, 1H, exchanged with deuterium oxide), 4.63 (s,
1H), 7.53-7.63 (m, 4H), 7.82 (t, 1H, J=7.6), 8.33-8.36 (m, 2H),
+
2H), 8.83 (d, 1H, J=7.6); ms (EI): m/z 569 (5, M ), 372 (60), 344
(45), 330 (30), 316 (35), 274 (53), 200 (50), 172 (50), 153 (60),
13
8.39 (d, 1H, J=8.9), 8.51 (d, 1H, J=8.9); C-nmr: δ 17.1, 26.9,
76.4, 85.6, 102.8, 110.4, 122.5, 123.2, 124.7, 125.8, 127.2, 128.9,
130.0, 130.9, 131.1, 132.3, 143.4, 157.2, 162.5, 191.1; ms (ESI):
+
NO Na hrms Calcd. 592.1942 (MNa );
139 (100); For C H
33 31
Found 592.1937.
8
+
m/z 388 (M+H) .
Anal. Calcd for C H NO : C, 71.31; H, 4.42; N, 3.62.
23 17 5
Found: C, 71.38; H, 4.46; N, 3.69.
Competition of the Tested Compounds with Dimethylsulfoxide
for Hydroxyl Radicals.
3+
The hydroxyl radicals generated by the Fe /ascorbic acid sys-
The fractions eluted next gave yellow crystals of compound 16
(117 mg, 54%), m.p. 238-240 °C (from ethanol); ir: 3430, 1710,
tem were detected by the determination of formaldehyde pro-
duced from the oxidation of dimethylsulfoxide [26]. The reaction
3+
mixture contained EDTA (0.1mM), Fe (167 µM, as a 1:2 mix-
-1
1
1570 cm ; H-nmr: δ 1.57 (s, 6H), 3.23 (s, 2H, exchanged with
deuterium oxide), 4.00 (d, 1H, J=5.08), 5.33 (d, 1H, J=5. 08),
7.51-7.62 (m, 4H), 7.74 (t, 1H, J=7.6), 8.28 (d, 1H, J=7.6), 8.45-
ture with EDTA), dimethylsulfoxide (33mM), in phosphate
buffer (50 mM, pH 7.4), the tested compounds (final concentra-
tion 1mM-final volume of the samples 1 mL). 150 µL of ascorbic
acid (10 mM in phosphate buffer was added at the end in order to
have the reaction started). The mixture was incubated at 37 °C for
30 min. The reaction was stopped by the addition of 250 µL
trichloroacetic acid (17.5 % w/v) and the formaldehyde formed
was detected spectrophotometrically at 412 nm by the method of
Nash [35].
13
8.48 (m, 2H), 8.81 (d, 1H, J=7.6); C-nmr: δ 21.5, 25.3, 61.4,
71.2, 80.0, 105.8, 109.6, 121.4, 122.6, 122.9, 123.3, 126.3, 128.4,
129.0, 129.1, 129.6, 130.7, 145.6, 151.5, 152.8, 160.4; ms (ESI):
+
+
m/z 390 (M+H) , 412 (M+Na) .
Anal. Calcd for C NO : C, 70.94; H, 4.92; N, 3.60.
Found: C, 70.59; H, 4.92; N, 3.83.
H
23 19
5
Reaction of Compound 16 with Compound 1 2. Preparation of
Compounds 17 , 17 , 18.
I
II
Interaction of the Synthesized Compounds with DPPH [34,36].
To a solution of compound 16 (91 mg, 0.23 mmole) in a mix-
ture of dry dichloromethane (4 mL) and dry pyridine (1 mL)
compound 12 (203 mg, 0.94 mmole) was added at 0 °C and the
mixture was stirred at room temperature for 18 hours under nitro-
gen. The mixture was concentrated in a rotary evaporator and the
residue was treated with water (10 mL) and extracted with chlo-
roform (10 mL). The organic layer was washed with water (5
mL), then with brine (5 mL), was dried over anhydrous sodium
sulfate and was concentrated. The residue was subjected to col-
umn chromatography (silica gel, dichloromethane/ethyl acetate
30:1). The fractions eluted first gave yellow crystals of com-
To a solution of DPPH (0.1- 0.5 mM) in absolute ethanol, an
equal volume of the compounds dissolved in ethanol was added
(0.1 mM). A control solution containing ethanol was also used.
After 20 and 60 min at room temperature, absorbance was
recorded at 517 nm (Table 1).
Soybean Lipoxygenase Inhibition [36].
The tested compounds, dissolved in 60% aqueous ethanol
(final concentration 0.1 mM), were incubated at room tempera-
ture with sodium linoleate (0.1 mM) and 0.15 mL of enzyme
4
pound 17 (16 mg, 9%), m.p. 254-256 °C (from ethanol); ir
I
(dichloromethane): 3035, 1780, 1740, 1730, 1690, 1670, 1600
solution (1/10 w/v in saline). The conversion of sodium
linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded
and compared with an appropriate standard inhibitor (nordihy-
droguaretic acid 0.1mM, 83.7%).
-1
cm ; H-nmr: δ 0.99 (s, 3H), 1.06 (s, 3H), 1.09 (s, 3H), 1.12 (s,
1
3H), 1.15 (s, 3H), 1.55 (s, 3H), 1.61 (s, 3H), 1.69 (s, 3H), 1.84-

Jul-Aug 2004
Synthesis and Biological Evaluation of Benzo[7,8]chromeno[5,6-b][1,4]oxazin-3-ones
611
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Acknowledgements.
The authors thank very much Prof E. DeClercq, Katholique
University of Leuven, Belgium, for the anti-HIV screening tests.
D. R. Gautam thanks State Scholarships Foundation (IKY),
Greece for financial support and Tribhuvan University,
Kathmandu, Nepal for granting him the PhD study leave.
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